Metagenomic sequencing enables accurate pathogen and antimicrobial susceptibility profiling in complicated UTIs in approximately four hours. Metagenomic sequencing enables accurate pathogen and antimicrobial susceptibility profiling in complicated UTIs in approximately four hours

Urinary tract infections (UTIs) affect 405 million people worldwide. Current diagnostics rely on cultures, which can take 2 to 4 days. This study evaluates eleven culture-independent methods for sample preparation from 78 complicated UTI patients, followed by real-time nanopore sequencing and data analysis. The metagenomic results are highly consistent with culture-based clinical routines (MALDI-TOF/VITEK-2). The optimized method demonstrated an accuracy score of 99% (100/101) for pathogen identification and 90% (589/ 653) for antimicrobial susceptibility profiling with 95% specificity. The method’s robustness is highlighted by its ability to accurately identify pathogens with as few as 32 bacterial cells/µL and a low bacterial-to-host cell ratio limit of 0.5. Additionally, mNGS identified 13 pathogens that routine diagnostics missed, which were subsequently confirmed by Vivalytic or PCR. This method is up to 30% more economical than published studies and commercial kits. DNA yield and flow cytometry can be used for pre-screening to reduce costs, which is crucial for clinical adoption. This research highlights the rapid diagnosis of clinical UTIs using a cost-effective and scalable method that requires around four hours from sample collection to informed decision-making. Furthermore, it aims to improve antimicrobial and diagnostic stewardship by reducing empirical treatment and ensuring more judicious antibiotic use.